The composition of a plant cell wall is complex and variable. Polysaccharides are mainly found in the form of long chains of cellulose (the main structural component of the plant cell wall), hemicellulose (comprising various .beta.-xylan chains) and pectin. The occurrence, distribution and structural features of plant cell wall polysaccharides are determined by (1) plant species; (2) variety; (3) tissue type, (4) growth conditions; (5) ageing and (6) processing of plant material prior to feeding.
Basic differences exist between monocotyledons (e.g. cereals and grasses) and dicotyledons (e.g. clover, rapeseed and soybean) and between the seed and vegetative parts of the plant (Chesson, 1987; Carre and Brillouet, 1986). Monocotyledons are characterized by the presence of an arabinoxylan complex as the major hemicellulose backbone. The main structure of hemicellulose in dicotyledons is a xyloglucan complex. Moreover, higher pectin concentrations are found in dicotyledons than in monocotyledons. Seeds are generally very high in pectic substances but relatively low in cellulosic material.
A cross-sectional diagram of a plant cell is depicted in FIG. 1. Three more or less interacting polysaccharide structures can be distinguished in the cell wall:
(1) The middle lamella forms the exterior cell wall. It also serves as the point of attachment for the individual cells to one another within the plant tissue matrix. The middle lamella consists primarily of calcium salts of highly esterified pectins; PA1 (2) The primary wall is situated just inside the middle lamella. It is a well-organized structure of cellulose microfibrils embedded in an amorphous matrix of pectin, hemicellulose, phenolic esters and proteins; PA1 (3) The secondary wall is formed as the plant matures. During the plant's growth and ageing phase, cellulose microfibrils, hemicellulose and lignin are deposited.
The primary cell wall of mature, metabolically active plant cells (e.g. mesophyll and epidermis) is more susceptible to enzymatic hydrolysis than the secondary cell wall, which by this stage, has become highly lignified.
There is a high degree of interaction between cellulose, hemicellulose and pectin in the cell wall. The enzymatic degradation of these rather intensively crosslinked polysaccharide structures is not a simple process. At least five different enzymes are needed to completely break down an arabinoxylan, for example. The endo-cleavage is effected by the use of an endo-.beta.(1.fwdarw.4)-D-xylanase-Exo-(1.fwdarw.4)-D-xylanase liberates xylose units at the non-reducing end of the polysaccharide. Three other enzymes (.alpha.-glucuronidase, L-arabinofuranosidase and acetyl esterase) are used to attack substituents on the xylan backbone. The choice of the specific enzymes is of course dependent on the specific hemicellulose to be degraded (McCleary and Matheson, 1986).
For certain applications, however, complete degradation of the entire hemicellulose into monomers is not necessary or is not desirable. In the liquefaction of arabinoxylan, for example, one needs simply to cleave the main xylan backbone into shorter units. This may be achieved by the action of an endo-xylanase, which ultimately results in a mixture of xylose monomer units and oligomers such as xylobiose and xylotriose. These shorter subunits are then sufficiently soluble for the desired use.
Filamentous fungi are widely known for their capacity to secrete large amounts of a variety of hydrolytic enzymes such as .alpha.-amylases, proteases and amyloglucosidases and various plant cell wall degrading enzymes such as cellulases, hemicellulases, and pectinases. Among these, multiple xylan-degrading enzymes have been recognized, which have been shown to possess a variety of biochemical and physical properties. This heterogeneity in xylanase function allows for the selection of a xylanase of interest which is best suited for a desired application (see Wong et al. (1988), Woodward (1984) and Dekker and Richards (1977)).
Multiple xylanases of various molecular weights are known to be produced by micro-organisms such as Aspergillus niger, Clostridium thermocellum, Trichoderma reesei, Penicillium janthinellum, as well as species of Bacillus and Streptomyces.
On the contrary, in yeast no xylanase multiplicity has been observed. In three yeast genera, Trichosporon, Cryptococcus and Aureobasidium, only a single xylanase could be detected.
In nature, microbial xylanases are always produced together with other enzymes having polysaccharide-degrading activities, such as exo-arabinanase, acetyl esterase and cellulases. For some applications, these enzyme activities are not needed or are unwanted.
It is known that fermentation conditions may be varied to favor the production of an enzyme of interest. It is also known that the cloning of the gene encoding the desired enzyme and overexpressing it in its natural host, or other compatible expression host will specifically enhance the production of the enzyme of interest. This latter method is particularly useful if the enzyme of interest is to be obtained in a form which is free of undesired enzyme activity.
The expression of recombinant bacterial xylanase has been previously described in European Patent Application 121.138. The gene encoding the bacterial xylanase was isolated from Bacillus chromosomal DNA and brought to expression in an E. coli host. However, E. coli expression hosts are, in some instances, considered to be unsafe for the production of proteins by recombinant DNA methods due to their production of unacceptable by-products such as toxins.
Since bacterial genes contain no introns, one is confronted with few problems in cloning and expressing such genes in prokaryotic hosts. On the other hand, the expression of eukaryotic genes is not always so straightforward. It is well known that genes isolated from eukaryotic strains contain introns. This inherently introduces complications in the cloning and expression of these genes, should a prokaryotic host be preferred.
Furthermore, certain differences exist, in general, between the physical characteristics of xylanases of fungal origin and those from bacteria. In general, fungal xylanases have a pH optimum in the range of between pH 3.5-5.5 as compared to bacterial xylanases which generally have a pH optimum in the range of pH 5.0-7.0. Fungal xylanases also generally have a broader pH stability range (pH 3-10) than do their bacterial counterparts (pH 5.0-7.5). Fungal xylanases generally have a temperature optimum of about 50.degree. C. Bacterial xylanases generally have a temperature optimum between 50.degree. C. and 70.degree. C. For a further discussion of the physical characteristics of xylanases see Wong et al. (1988), Woodward (1984) and Dekker and Richards (1977).
Thus, it is clear that bacterial xylanases are less suitable for use in, for example, processes requiring lower pH conditions. In other instances, bacterial xylanases are too thermostable for certain applications such as the lagering of beer (see European Patent No. 227,159).
Accordingly, it would be of great importance to obtain genes encoding xylan-degrading enzymes of fungal origin which may be brought to expression in other, high-producing microbial expression hosts.